U.S. patent number 10,227,372 [Application Number 15/517,313] was granted by the patent office on 2019-03-12 for bola-amphiphilic compounds and their uses for biomedical applications.
This patent grant is currently assigned to INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), UNIVERSITE DE BORDEAUX. The grantee listed for this patent is INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), UNIVERSITE DE BORDEAUX. Invention is credited to Ananda Appavoo, Philippe Barthelemy, Olivier Chassande, Camille Ehret, Laurent Latxague, Michael Ramin.
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United States Patent |
10,227,372 |
Barthelemy , et al. |
March 12, 2019 |
Bola-amphiphilic compounds and their uses for biomedical
applications
Abstract
The invention relates to bola-amphiphilic compounds and their
uses for biomedical application. The invention particularly relates
to the use of bola-amphiphilic compounds for providing low
molecular weight gels (LMWG), useful, in particular, as culture
media for animal or human cells, or as biocompatible material for
biomedical applications.
Inventors: |
Barthelemy; Philippe (Merignac,
FR), Ramin; Michael (Bordeaux, FR),
Latxague; Laurent (Saint-Medard-en-Jalles, FR),
Appavoo; Ananda (Bordeaux, FR), Chassande;
Olivier (Pessac, FR), Ehret; Camille (Haguenau,
FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITE DE BORDEAUX
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM) |
Bordeaux
Paris |
N/A
N/A |
FR
FR |
|
|
Assignee: |
UNIVERSITE DE BORDEAUX
(Bordeaux, FR)
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM) (Paris, FR)
|
Family
ID: |
51830251 |
Appl.
No.: |
15/517,313 |
Filed: |
October 6, 2015 |
PCT
Filed: |
October 06, 2015 |
PCT No.: |
PCT/EP2015/073072 |
371(c)(1),(2),(4) Date: |
April 06, 2017 |
PCT
Pub. No.: |
WO2016/055493 |
PCT
Pub. Date: |
April 14, 2016 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20170240583 A1 |
Aug 24, 2017 |
|
Foreign Application Priority Data
|
|
|
|
|
Oct 6, 2014 [EP] |
|
|
14290302 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H
1/00 (20130101); C12N 5/0068 (20130101); C07H
19/06 (20130101); C07H 19/073 (20130101); A61K
47/26 (20130101); A61K 9/06 (20130101); C12N
2539/00 (20130101); C12N 2533/20 (20130101); C12N
2513/00 (20130101) |
Current International
Class: |
C07H
19/06 (20060101); C12N 5/00 (20060101); A61K
47/26 (20060101); C07H 19/073 (20060101); A61K
9/06 (20060101); C07H 1/00 (20060101) |
Foreign Patent Documents
Other References
Tiwari, P. et al. "Efficient Acetylation of Carbohydrates Promoted
by Imidazole" Eur. J. Org. Chem. 2005, 4265-4270 (Year: 2005).
cited by examiner .
Godeau, G. et al. "Glycosyl-nucleoside-lipid based supramolecular
assembly as a nanostructured material with nucleic acid delivery
capabilities" Chem. Commun., 2009, 5127-5129 (Year: 2009). cited by
examiner .
Barthelemy, P. et al. "Glycosyl-Nucleoside Lipids as
Low-Molecular-Weight Gelators" Langmuir 2009, 25(15), 8447-8450
(Year: 2009). cited by examiner .
Iwaura, R. et al. "Oligonucleotide-Templated Self-Assembly of
Nucleotide Bolaamphiphiles: DNA-Like Nanofibers Edged by a
Double-Helical Arrangement of A-T Base Pairs" Angew. Chem. Int. Ed.
2003, 42, No. 9 (Year: 2003). cited by examiner .
Latxague, L. et al. "Glycosyl-Nucleolipids as New Bioinspired
Annphiphiles" Molecules 2013, 18, 12241-12263 (Year: 2013). cited
by examiner .
Guilhem Godea et al.,"Glycosyl-nucleoside-lipid based
supramolecular assembly as a nanostructured material with nucleic
acid delivery capabilities", Chemical Communication, p. 5127-5129,
No. 34 (Jan. 2009). cited by applicant .
Sophia Ziane et al.,"A thermosensitive low molecular weight
hydrogel as scaffold for tissue engineering", European cells &
materials, pp. 147-160 (Jan. 2012). cited by applicant .
Laurent Latxague et al.,"Glycosyl-Nucleolipids as New Bioinspired
Amphiphiles", Molecules, pp. 12241-12263, vol. 18, No. 10 (Jan.
2013). cited by applicant .
Laurent Latxague et al.,"Glycosylated nucleoside lipid promotes the
liposome internalization in stem cells", Chemical Communications,
pp. 12598-12600, vol. 47, No. 47 (Jan. 2011). cited by applicant
.
Nuraje Nurxat et al.,"Bolaamphiphilic molecules: Assembly and
application" Progress in Polymer Science, pp. 302-343, vol. 38. No.
2 (Sep. 2012). cited by applicant.
|
Primary Examiner: Rosenthal; Andrew S
Attorney, Agent or Firm: Browdy and Neimark, PLLC
Claims
The invention claimed is:
1. A compound of formula (I) R.sub.1--X-A-X--R.sub.2 (I) in which X
is oxygen, --NH--C(O)--NH-- or --C(O)--NH-- A is a C.sub.4-C.sub.30
hydrocarbon chain, linear or branched, saturated or unsaturated,
which is unsubstituted or substituted by one or more
C.sub.1-C.sub.12 linear or branched alkyl groups, or A represents a
C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated
or unsaturated, which is partially or completely halogenated;
R.sub.1 and R.sub.2, identical or different, represent
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.-
2).sub.p--R.sub.5--R.sub.6 in which n, m and p, identical or
different, are 0 to 10; R.sub.3 represents a heteroaryl group
comprising 1 to 4 oxygen or nitrogen atom(s); R.sub.4 represents a
nucleosidyl group; R.sub.5 represents a heteroaryl group comprising
1 to 4 heteroatom(s); R.sub.6 represents a residue of a cyclic
carbohydrate or a derivative of the said carbohydrate.
2. A compound of formula (I) according to claim 1, in which the
carbohydrate derivative of R.sub.6 is selected from an
oligosaccharide-type glycan deriving from a cyclic carbohydrate, a
N-acyl derivative of said cyclic carbohydrate and a protected
derivative of said cyclic carbohydrate.
3. A compound of formula (I) according to claim 1, in which at
least one of the following conditions is fulfilled: n=m=p=1;
R.sub.3 and R.sub.5, identical or different, represent a heteroaryl
group containing 1 to 4 nitrogen atoms selected from the group
consisting of pyrazole, triazole, tetrazole and imidazole; R.sub.4
represents a nucleosidyl group selected from adenosine,
deoxyadenosine, guanosine, deoxyguanosine, thymidine,
deoxythymidine, uridine, deoxyuridine, cytidine and deoxycytidine;
R.sub.6 represents a residue of a cyclic carbohydrate selected from
D-glucopyranose, D-galactopyranose, D-mannopyranose,
D-fructopyranose D-ribofuranose, N-acetylglucosamine,
N-acetylgalactosamine, N-acetylmannosamine and sialic acid or a
protected derivative thereof.
4. A compound of formula (I) according to claim 1, in which: A
represents a C.sub.12 saturated hydrocarbon chain; R.sub.1 is
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6; R.sub.2 is
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6; n=m=p=1; R.sub.3 is a triazole group; R.sub.4 is
thymidine; R.sub.5 is a triazole group, and R.sub.6 is
D-glucopyranosyl or 2,3,4,6 tetra-O-protected-glucopyranosyl.
5. A compound of formula (I) according to claim 1, in which: A
represents a C.sub.10 hydrocarbon chain which is partially
fluorinated; R.sub.1 and R.sub.2 are
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6; n=m=p=1; R.sub.3 is a triazole group; R.sub.4 is
thymidine; R.sub.5 is a triazole group, and R.sub.6 is
D-glucopyranosyl or 2,3,4,6 tetra-O-protected-glucopyranosyl.
6. A compound of formula (I) according to claim 1, in which R.sub.5
is covalently linked to R.sub.4 via --(CH.sub.2)p- to the carbon
atom at position 5' of the ribose or deoxyribose ring of the
nucleosidyl group, or, alternatively, R.sub.5 is covalently linked
to R.sub.4 via --(CH.sub.2)p- to the oxygen atom at position 3' of
the ribose or deoxyribose ring of the nucleosidyl group.
7. A compound of formula (I) according to claim 1, which is
selected from the group consisting of:
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((.b-
eta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine,
1,12-bis-dodecanyl-5'-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-5'-deoxy-3'-O-
-1-((b-D-glucopyranoside)-1-H-1,2,3-triazol-4-yl)methyl thymidine,
1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5'-[4-(oxymeth-
yl)-1H-1,2,3-triazol-1-yl]-N-3-[1-((b-D-glucopyranoside)-1H-1,2,3-triazol--
4-yl)methyl]-5'-deoxythymidine,
1,12-bis-dodecanyl-5'-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3-
,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methy-
l]-5'-deoxythymidine,
1,12-bis-dodecanyl-5'-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((.be-
ta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine,
1,14-bis-tetradecanyl-5'-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-(-
(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)m-
ethyl]-5'-deoxy thymidine, and
1,14-bis-tetradecanyl-5'-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-(-
(.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine.
8. A process for preparing a compound of formula (I) according to
claim 1 in which X is oxygen, comprising the following steps:
reacting an alkane-diol or a fluoroalkane-diol of formula
R.sub.1--O-A-O--R.sub.2 at both ends with an alkylating agent of
formula R--(CH.sub.2).sub.q--C.ident.CH, where q is 1 or 2 and R is
a halide, in particular chloride or bromide, reacting the resulting
compound with the 5'-azido derivative of R.sub.4, reacting the
purine or pyrimidine base or universal base moiety of R.sub.4 in
the resulting compound with an alkylating agent of formula
R--(CH.sub.2).sub.q--C.ident.CH, where q and R is as defined above,
in order to obtain the N-alkylated derivative of the purine or
pyrimidine base or universal base of R.sub.4, and reacting the
resulting compound with the 1-azido derivative of R.sub.6
(click-reaction).
9. A process for preparing a compound of formula (I) according to
claim 1 in which X is --NH--C(O)--NH--, comprising the following
steps: reacting a diisocyanatoalkane of formula OCN-A-NCO with
NH.sub.2--CH.sub.2--C.ident.CH (propargylamine) or any primary
amine containing a terminal alkyne group in order to obtain a
compound of formula (II)
CH.ident.C--CH.sub.2--NH--C(O)--NH-A-NH--C(O)--NH--CH.sub.2--C.ident.CH
(II), reacting a purine or pyrimidine base or a universal base with
an alkylating agent of formula R--(CH.sub.2).sub.qC.dbd.CH, where q
is 1 or 2 and R is a halide, in order to obtain the N-alkylated
derivative of the purine or pyrimidine base or universal base of
R.sub.4, reacting the resulting compound with the 1-azido
derivative of R.sub.6 (click-reaction), converting the 5'--OH group
on the ribose or deoxyribose moiety of R.sub.4 into an azide, and
subjecting the resulting compound to a second click-reaction with a
compound of formula (II).
10. A hydrogel formed from one or more compound(s) of formula (I)
according to claim 1.
11. A hydrogel according to claim 10, characterized in that they
are formed from an aqueous solution of compounds of formula (I)
containing 0.1 to 10 wt % of compound of formula (I).
12. A hydrogel according to claim 10, characterized in that said
hydrogel comprises one or more compound(s) of formula (I), or a
mixture of one or more compound(s) of formula (I) and one or more
glycosyl-nucleoside(s)-lipid(s) (GNL) and/or
glycosyl-nucleoside(s)-fluorolipid(s) (GNF), or else a mixture of
one or more compound(s) of formula (I) with one or more protein(s)
or glycoprotein(s), where said mixture contains, optionally, one or
more GNL or GNF.
13. In a cell culture media containing a hydrogel, the improvement
wherein said hydrogel is one according to claim 10.
14. A cell culture media according to claim 13, where the cells are
eukaryotic cells.
15. A cell culture media according to claim 13, where the cells are
stem cells.
16. A hydrogel according to claim 10 for use as a biocompatible
material for tissue engineering, cellular therapy or prevention of
adhesion formation after abdomino-pelvic surgery.
17. A hydrogel according to claim 10 for use as a biocompatible
material for controlled-release delivery of an active ingredient
which is entrapped into the gel.
18. The process of claim 8 wherein R is selected from chlorine or
bromine.
19. The process of claim 9 wherein R is selected from chlorine or
bromine.
Description
The invention relates to bola-amphiphilic compounds and their uses
for biomedical applications.
The invention particularly relates to the use of bola-amphiphilic
compounds for providing low molecular weight gels (LMWG), useful,
in particular, as culture media for cells, in particular isolated
stem cells.
Hydrogels which are non-toxic, easy to use, cytocompatible,
injectable and degradable are valuable biomaterials as cell-culture
media.
The development of biocompatible artificial matrixes that can be
used for stem cell culture remains a great challenge in cell
culture engineering and/or regenerative medicine. Most of gel
scaffold developed so far involve polymeric materials derived from
either natural sources or chemical synthesis. However, polymers
often suffer from several limitations, including poor
biocompatibility, toxicity, biodegradability, pro-inflammatory
activity etc. Alternatively, small-molecule based hydrogels are
currently emerging as a new powerful tool for regenerative medicine
strategy capable of restoring biological and mechanical properties
and/or function.
The invention thus relates to new bola-amphiphilic compounds
derived from nucleolipids and to a new generation of low molecular
weight gels (LMWG) which are suitable for cell culture, in
particular for stem cell culture. Bola-amphiphile based hydrogel
matrixes exhibit the following requested properties: non-toxicity,
easy to handle, injectability, and biocompatible rheology
(thixotropic behavior).
Bola-amphiphiles are composed of one or two hydrophobic chains
covalently linked at both ends to hydrophilic head groups. This
type of molecular architecture, which can be found in
archaebacteria membranes, have been used in numerous applications
which range from nanomaterial synthesis to drug or gene
delivery.
The use of glycosyl-nucleosides-lipids (GNL) and
glycosyl-nucleosides-fluorolipids (GNF) for forming LMWG has been
reported.
GNL have been reported as being useful for promoting
internalization of GNL based liposomes into stem cells (L. Latxague
et al., Chem. Commun., 2011, 47, 12598-12600).
GNL supramolecular assemblies for nucleic acid delivery and
GNL-based gels were studied in G. Godeau et al., Chem. Commun.,
2009, 1-3.
GNF-based gels were reported as being only compatible with stem
cell cultures where the stem cells formed aggregates, and clearly
not compatible with the survival and growth of isolated stem cells
(S. Ziane et al., Eur. Cells and Math., 2012, 23, 147-160).
It has now been found that new glycosylated nucleoside based
bola-amphiphile (GNBA) compounds exhibit particular physicochemical
properties which render them particularly suitable for providing
low molecular weight gels.
The invention thus relates to a compound of formula (I)
R.sub.1--X-A-X--R.sub.2 (I)
in which X is oxygen, --NH--C(O)--NH-- or --C(O)--NH-- A is a
C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated
or unsaturated, which is unsubstituted or substituted by one or
more C.sub.1-C.sub.12 linear or branched alkyl groups, or A
represents a C.sub.4-C.sub.30 hydrocarbon chain, linear or
branched, saturated or unsaturated, which is partially or
completely halogenated; R.sub.1 and R.sub.2, identical or
different, represent hydrogen or
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6
in which n, m and p, identical or different, are 0 to 10; R.sub.3
represents a heteroaryl group comprising 1 to 4 oxygen or nitrogen
atom(s); R.sub.4 represents a nucleosidyl group or does not exist;
R.sub.5 represents a heteroaryl group comprising 1 to 4
heteroatom(s); R.sub.6 represents a residue of a cyclic
carbohydrate or a derivative of the said carbohydrate;
provided that R.sub.1 and R.sub.2 are not simultaneously
hydrogen.
Preferably, A is a C.sub.4-C.sub.18 hydrocarbon chain, more
preferably a C.sub.12 or C.sub.14 hydrocarbon chain, linear or
branched, saturated or unsaturated, which is unsubstituted or
substituted by one or more C.sub.1-C.sub.12 linear or branched
alkyl groups;
Alternatively, A is a C.sub.4-C.sub.18 hydrocarbon chain, more
preferably a C.sub.10 or C.sub.12 hydrocarbon chain linear or
branched, saturated or unsaturated, which is partially or
completely halogenated.
Preferably, the invention relates to compound of formula (I) in
which R.sub.4 represents a nucleosidyl group.
Preferably, R.sub.1 and R.sub.2, identical or different, represent
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6.
According to a preferred embodiment, the invention relates to a
compound of formula (I) R.sub.1--X-A-X--R.sub.2 (I)
in which X is oxygen, --NH--C(O)--NH-- or --C(O)--NH-- A is a
C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated
or unsaturated, which is unsubstituted or substituted by one or
more C.sub.1-C.sub.12 linear or branched alkyl groups, or A
represents a C.sub.4-C.sub.30 hydrocarbon chain, linear or
branched, saturated or unsaturated, which is partially or
completely halogenated; R.sub.1 and R.sub.2, identical or
different, represent
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6
in which n, m and p, identical or different, are 0 to 10; R.sub.3
represents a heteroaryl group comprising 1 to 4 oxygen or nitrogen
atom(s); R.sub.4 represents a nucleosidyl group; R.sub.5 represents
a heteroaryl group comprising 1 to 4 heteroatom(s); R.sub.6
represents a residue of a cyclic carbohydrate or a derivative of
the said carbohydrate.
R.sub.5 can be covalently linked to R.sub.4 via
--(CH.sub.2).sub.p-- to the carbon atom at position 5' of the
ribose or deoxyribose ring of the nucleoside, or, alternatively, to
the oxygen atom at position 3' of the ribose or deoxyribose ring of
the nucleoside.
<<Heteroaryl group containing 1 to 4 oxygen or nitrogen
atoms>> refers to a monocyclic or bicyclic, aromatic or
partially unsaturated, carbocyclic group containing 5 to 12 atoms,
interrupted by 1 to 4 oxygen or nitrogen atoms, which can be, for
example, selected from furane, pyrrole, oxazole, oxadiazole,
isoxazole, pyrazole, triazole, tetrazole, imidazole, pyridine,
pyrimidine, pyridazine, pyrazine, benzofurane, indole, quinoleine,
isoquinoleine, chromane, naphtyridine or benzodiazine groups,
triazole being preferred.
<<Hydrocarbon chain, which is partially or completely
halogenated>> refers to a saturated or unsaturated alkyl
chain in which some or all hydrogen atoms are replaced by halogen
atoms, such as fluorine, iodine, chlorine or bromine, fluorine
being preferred.
<<Nucleosidyl group>> refers to a group consisting of a
ribose or deoxyribose moiety which is linked to a purine or
pyrimidine base, or to derivatives of said purine or pyrimidine
base, or to a non-natural mono or bicyclic heterocyclic base, all
said bases being optionally substituted.
The purine base can be, for example, selected from the group
consisting of adenine, guanine and hypoxanthine.
The pyrimidine base can be, for example, selected from the group
consisting of thymine, uracile and cytosine, thymine being
preferred.
By <<non-natural mono- or bicycle heterocyclic base>>
is meant a universal base, such as, for example, 3-nitropyrrole,
4-nitroimidazole or 5-nitroindole.
By <<optionally substituted>> is meant that the purine
or pyrimidine base, or the non-natural heterocyclic base can be
substituted by at least one substituent chosen, for example, from a
halogen, an amino group, a carboxy group, a carbonyl group, a
carbonylamino group, a hydroxy, azido, cyano, alkyl, cycloalkyl,
perfluoroalkyl, alkyloxy (for example, methoxy), oxycarbonyl,
vinyl, ethynyl, propynyl, acyl group etc.
<<Residue of a cyclic carbohydrate or a derivative of the
said carbohydrate>> refers to a residue of a 5- or 6-membered
osidic cycle, which can be, for instance, selected from
D-glucopyranose, D-galactopyranose, D-mannopyranose,
D-fructopyranose or D-ribofuranose, or oligosaccharide-type glycans
deriving therefrom, as well as their N-acyl derivatives, in
particular their N-acetyl derivatives, such as, for instance,
N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine or
sialic acid, or a protected derivative thereof, such as an O-acyl
derivative, in particular an O-acetyl derivative, D-glucopyranose
being preferred.
In particular, the invention relates to compounds of formula (I),
in which at least one of the following conditions is fulfilled:
n=m=p=1
R.sub.3 and R.sub.5, identical or different, represent a heteroaryl
group containing 1 to 4 nitrogen atoms selected from the group
consisting of pyrazole, triazole, tetrazole and imidazole;
R.sub.4 represents a nucleosidyl group selected from adenosine,
deoxyadenosine, guanosine, deoxyguanosine, thymidine,
deoxythymidine, uridine, deoxyuridine, cytidine and
deoxycytidine.
R.sub.6 represents a residue of a cyclic carbohydrate selected from
D-glucopyranose, D-galactopyranose, D-mannopyranose,
D-fructopyranose, D-ribofuranose, N-acetylglucosamine,
N-acetylgalactosamine, N-acetylmannosamine and sialic acid or a
protected derivative thereof.
Preferred compounds of formula (I) are those in which X is
oxygen.
Alternatively, compounds of formula (I) of interest are those in
which X is --NH--C(O)--NH-- or --C(O)--NH--.
Preferred compounds of formula (I) are those in which:
A represents a C.sub.12 or C.sub.14 saturated hydrocarbon
chain;
R.sub.1 is hydrogen, or
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6;
R.sub.2 is
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.n--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6;
n=m=p=1;
R.sub.3 is a triazole group;
R.sub.4 is thymidine;
R.sub.5 is a triazole group, and
R.sub.6 is .beta.-D-glucopyranosyl or 2,3,4,6
tetra-O-protected-glucopyranosyl, preferably 2,3,4,6
tetra-O-acetyl-glucopyranosyl.
Other preferred compounds are those in which
A represents a C.sub.10 hydrocarbon chain which is partially
fluorinated;
R.sub.1 and R.sub.2 are
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6;
n=m=p=1;
R.sub.3 is a triazole group;
R.sub.4 is thymidine;
R.sub.5 is a triazole group, and
R.sub.6 is .beta.-D-glucopyranosyl or 2,3,4,6
tetra-O-protected-glucopyranosyl, preferably 2,3,4,6
tetra-O-acetyl-glucopyranosyl.
According to a preferred embodiment, R.sub.5 is covalently linked
to R.sub.4 via --(CH.sub.2)p- to the carbon atom at position 5' of
the ribose or deoxyribose ring of the nucleosidyl group.
Alternatively, R.sub.5 is covalently linked to R.sub.4 via
--(CH.sub.2)p- to the oxygen atom at position 3' of the ribose or
deoxyribose ring of the nucleosidyl group.
Particularly preferred compounds of formula (I) are:
5'-[4-(12-hydroxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5'-deoxy-N3--
(1-((.beta.-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl
thymidine,
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((.b-
eta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine,
1,12-bis-dodecanyl-5'-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-5'-deoxy-3'-O-
-1-((.beta.-D-glucopyranoside)-1-H-1,2,3-triazol-4-yl)methyl
thymidine,
1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5'-[4-(oxymeth-
yl)-1H-1,2,3-triazol-1-yl]-N-3-[1-((.beta.-D-glucopyranoside)-1H-1,2,3-tri-
azol-4-yl)methyl]-5'-deoxythymidine,
1,12-bis-dodecanyl-5'-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3-
,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methy-
l]-5'-deoxythymidine,
1,12-bis-dodecanyl-5'-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((.be-
ta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine,
1,14-bis-tetradecanyl-5'-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-(-
(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)m-
ethyl]-5'-deoxy thymidine, and
1,14-bis-tetradecanyl-5'-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-(-
(.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine.
The compounds of formula (I) in which X is oxygen can be obtained
by a process comprising the following steps: reacting an
alkane-diol or a fluoroalkane-diol of formula
R.sub.1--O-A-O--R.sub.2 at both ends with an alkylating agent of
formula R--(CH.sub.2).sub.q--C.ident.CH, where q is 1 or 2 and R is
an halide, in particular chloride or bromide, reacting the
resulting compound with the 5'-azido derivative of R.sub.4,
reacting the purine or pyrimidine base or universal base moiety of
R.sub.4 in the resulting compound with an alkylating agent of
formula R--(CH.sub.2).sub.q--C.ident.CH, where q and R are as
defined above, in order to obtain the N-alkylated derivative of the
purine or pyrimidine base or universal base of R.sub.4, and
reacting the resulting compound with the 1-azido derivative of
R.sub.6 (click-reaction).
In order to obtain compounds of formula (I) in which X is oxygen
and R.sub.3 and/or R.sub.5 are not triazoles, the skilled person is
able to choose the appropriate cycloaddition reactions, such as
Diels-Alder reactions, in order to obtain the desired R.sub.3
and/or R.sub.5 heterocycles.
The following reaction conditions are preferred: the alkylating
reactions are performed in the presence of sodium hydride; the
click reaction is carried out with a 1:1 (v/v) mixture of water and
dichloromethane with vigorous stirring at 40.degree. C. or a 1:1
(v/v) mixture of water and THF at 60-65.degree. C. with 10%
CuSO.sub.4 and 20% ascorbic acid;
If desired, the synthesis of unsymmetrical compounds of formula (I)
where one of R.sub.1 and R.sub.2 represents hydrogen and the other
is
--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4-(CH.sub.2).sub.p---
R.sub.5--R.sub.6 can be undertaken starting with monoalkylated
starting materials clicked with the 5'-azido derivative of R.sub.4.
Subsequent reaction of the purine or pyrimidine base moiety present
in the resulting compound with an alkylating agent of formula
R--CH.sub.2--C.ident.CH followed by a second click reaction with
the 1-azido derivative of R.sub.6 affords the desired unsymmetrical
compound of formula (I).
The compounds of formula (I) in which X is --NH--C(O)--NH--
(bis-urea compounds) can be obtained by a process comprising the
following steps: reacting a diisocyanatoalkane of formula OCN-A-NCO
with NH.sub.2--CH.sub.2--C.ident.CH (propargylamine) or any primary
amine containing a terminal alkyne group) in order to obtain a
compound of formula (II)
CH.ident.C--CH.sub.2--NH--C(O)--NH-A-NH--C(O)--NH--CH.sub.2--C.ident.CH
(II), reacting a purine or pyrimidine base or a universal base with
an alkylating agent of formula R--(CH.sub.2).sub.q--C.ident.CH,
where q is 1 or 2 and R is an halide, preferably bromide or
chlorine, in order to obtain the N-alkylated derivative of the
purine or pyrimidine base or universal base of R.sub.4, reacting
the resulting compound with the 1-azido derivative of R.sub.6
(click-reaction), converting the 5'-OH group on the ribose or
deoxyribose moiety of R.sub.4 into an azide, and subjecting the
resulting compound to a second click-reaction with a compound of
formula (II).
The following reaction conditions are preferred: the alkylating
reaction of the purine or pyrimidine base or universal base with
R--CH.sub.2--C.ident.CH is carried out under basic conditions; the
first click-reaction is Cu(I) catalyzed and is carried out in a 1:1
(v/v) mixture of water and tert-butanol, preferably at 60.degree.
C., and preferably with 10% CuSO.sub.4 and 20% ascorbic acid; the
conversion of the 5'-OH group on the ribose or deoxyribose moiety
of R.sub.4 into an azide can be carried out by using an
organophosphorous compound, such as, for example,
triphenylphosphine (PPh.sub.3), a carbon halide such as, for
example, tetrabromide (CBr.sub.4) and sodium azide (NaN.sub.3).
The compounds of formula (I) in which X is --C(O)--NH-- (bis-amide
compounds) can be obtained by a process comprising the following
steps:
reacting an alkane diacid chloride prepared in situ from the
corresponding dicarboxylic acid of formula HOOC-A-COOH and thionyl
chloride, with NH.sub.2--CH.sub.2--C.ident.CH (propargylamine) or
any primary amine containing a terminal alkyne group in order to
obtain a compound of formula (III)
CH.ident.C--CH.sub.2--C(O)--NH-A-C(O)--NH--CH.sub.2--C.ident.CH
(III). The following steps are the same are those described above
for obtaining bis-urea compounds of formula (I).
The invention further relates to compounds of formula (IV)
R.sub.7--X-A-X--(CH.sub.2).sub.n--R.sub.3--(CH.sub.2).sub.m--R.sub.4--R.s-
ub.7 (IV)
In which X is --NH--C(O)--NH--; A is a C.sub.4-C.sub.30 hydrocarbon
chain, linear or branched, saturated or unsaturated, which is
unsubstituted or substituted by one or more C.sub.1-C.sub.12 linear
or branched alkyl groups, or A represents a C.sub.4-C.sub.30
hydrocarbon chain, linear or branched, saturated or unsaturated,
which is partially or completely halogenated; R.sub.3 represents a
heteroaryl group comprising 1 to 4 oxygen or nitrogen atom(s);
R.sub.4 represents a nucleosidyl group; R.sub.7 is the residue of
an alkylating agent of formula R--(CH.sub.2).sub.q--C.ident.CH
where q is 1 or 2 and R is an halide, which is linked by a covalent
bond with a nitrogen atom of the purine, pyrimidine or universal
base moiety of R.sub.4; n and m, identical or different, are 0 to
10.
R is preferably chloride or bromide, and q is preferably 1.
The compounds of formula (IV) are useful as synthesis intermediates
for obtaining the compounds of formula (I) in which X is
oxygen.
All the preferred features mentioned above for A, R.sub.3 and
R.sub.4 in formula (I) also apply to formula (IV).
Preferred compounds of formula (IV) are those in which n=m=1
R.sub.3 is a triazole group; R.sub.4 is thymidine; R.sub.7 is
propargyl.
In particular, compounds of formula (IV) of interest are:
5'-[4-((12-Propargyloxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5'-deo-
xy-N-3-propargylthymidine,
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazol-1-yl)]-5'-deoxy-N-3-
-propargyl thymidine, and
1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5'-[4-(oxymeth-
yl)-1H-1,2,3-triazol-1-yl]-N-3-propargyl-5'-deoxythymidine.
The invention further relates to compounds of formula (V)
R.sub.4--(CH.sub.2).sub.p--R.sub.5--R.sub.6 (V)
In which R.sub.4 represents a nucleosidyl group; R.sub.5 represents
a heteroaryl group comprising 1 to 4 heteroatom(s), which is linked
to R.sub.4 by a covalent bond to a nitrogen atom of the pyrimidine
base or of the purine base of the nucleosidyl group; R.sub.6
represents a residue of a cyclic carbohydrate or a derivative of
the said carbohydrate, which is optionally substituted; p is 0 to
10.
Preferably, R.sub.4 is a triazole group.
Preferably, R.sub.5 is thymidinyl, and R.sub.4 is linked to the
nitrogen in position 3 of R.sub.5.
By <<optionally substituted>> is meant that the cyclic
carbohydrate or derivative thereof can be substituted by at least
one substituent chosen, for example, from a halogen, an amino
group, a carboxy group, a carbonyl group, a carbonylamino group, a
hydroxy, azido, cyano, alkyl, cycloalkyl, perfluoroalkyl, alkyloxy
(for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl
group etc.
The compounds of formula (V) are useful as synthesis intermediates
for obtaining the compounds of formula (I) in which X is
--NH--C(O)--NH-- or --C(O)--NH--.
All the preferred features mentioned above for R.sub.4, R.sub.5 and
R.sub.7 in formula (I) also apply to formula (V).
Preferred compounds of formula (V) are those in which p=1 R.sub.3
is a triazole group; R.sub.5 is thymidine or R.sub.6 is a residue
of 2,3,4,6 tetra-O-protected-glucopyranosyl.
In particular, compounds of formula (V) of interest are:
5'-deoxy-N3-(1-(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-
-triazol-4-yl) thymidine, and
5'-Azido-N3-(1-(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-
-triazol-4-yl) thymidine.
The invention also relates to biocompatible hydrogels formed from
at least one compound of formula (I). In particular, these
hydrogels are formed from an aqueous solution of compounds of
formula (I) which can contain, for example, 0.1 to 10 wt % of
compound of formula (I), in particular 1 to 5 wt %.
The biocompatible hydrogels according to the invention can comprise
one or more compound(s) of formula (I), or a mixture of one or more
compound(s) of formula (I) and one or more
glycosyl-nucleoside(s)-lipid(s) (also called GNL) and/or
glycosyl-nucleoside(s)-fluorolipid(s) (also called GNF).
The biocompatible hydrogels according to the invention can also
contain a mixture of one or more compound(s) of formula (I) with
one or more protein(s) or glycoprotein(s), such as for example
collagen or hyaluronic acid, and optionally, one or more GNL or
GNF.
"Biocompatible" is understood as a material which is well tolerated
by the host organism and which does not cause any rejection, toxic
reaction, injury or harmful effect on the biological functions of
the host organism.
The biocompatible hydrogels according to the invention can be
prepared, for example, by hating the compounds of formula (I) until
dissolution, for example at a temperature of 40.degree. C. to
50.degree. C., and allowing the solution to cool gradually to room
temperature.
All the preferred features mentioned above for the compounds of
formula (I) also apply to the hydrogels formed therefrom.
The invention also relates to the use of hydrogels formed from
compounds of formula (I) as cell culture media, in particular as
culture media for animal or human cells.
The hydrogels according to the invention are particularly suitable
as culture media for eukaryotic cells, in particular for stem cell
culture. Indeed, it has been surprisingly found that these
hydrogels allow the culture of stem cells under isolated form,
while when using existing LMWG, such as those formed from GNF,
formation of clusters of stem cells was observed. The culture of
isolated eukaryotic cells, in particular stem cells, is highly
valuable and promising in view of the extensive research which is
currently carried out on stem cells. Advantageously, the hydrogels
formed from compounds of formula (I) possess particular
viscoelastic properties. Indeed, rheological studies on the
variation of the storage modulus G' (also called elastic modulus)
and the loss modulus G'' (also called viscous modulus) as a
function of the applied frequency were performed. G' describes the
amount of energy which is stored and released in each oscillation,
and G'' corresponds to the energy which is dissipated at heat.
Surprisingly, it has been found that the storage modulus G' is
higher than the loss modulus G'', which indicates the formation of
a stable gel. Without wishing to be bound by theory, it can be
hypothesized that this high elastic modulus plays a role in the
adhesion and proliferation of isolated stem cells.
In addition, it has been found that the hydrogel formed from
compounds of formula (I) possess a thixotropic behaviour, and thus
can be delivered by a syringe, thus allowing surgical use.
Actually, said hydrogel is able to regain its gel behaviour and
strength after a high strain is applied thereto.
In particular, the hydrogel according to the invention can be used
as biocompatible materials in the following biomedical
applications: Tissue engineering, for instance by injecting
gel-cell complexes: which can be used, for example, for bone
regeneration; Cellular therapy, for instance by injecting gel-cell
complexes in which the cells produce the active molecule(s)
Prevention of adhesion formation after abdomino-pelvic surgery, for
instance by injecting the gel which will remain at the application
location and act as a barrier between adjacent tissues.
Also, the biocompatible hydrogel according to the invention can be
used as biocompatible material for drug delivery, for instance: for
controlled-release delivery of an active ingredient which is
entrapped into the gel, allowing the active ingredient to diffuse
slowly near the injection site (topical administration, such as,
for instance, for chemotherapy) and/or in the blood flow; for
hosting delivery devices, such as, for instance for the
encapsulation of electrically stimulable devices.
The invention is non limitatively supported by the examples
below.
All commercially available reagents and solvents (Fluka,
Sigma-Aldrich, Alfa-Aesar) were used without further
purification.
For reactions requiring anhydrous conditions, dry solvents were
used (Sigma-Aldrich) under inert atmosphere (nitrogen or
argon).
Analytical thin layer chromatography (TLC) was performed on
pre-coated silica gel F.sub.254 plates with fluorescent indicator
(Merck). The detection of compounds was accomplished with UV light
(254 nm) and by subsequent spraying with 10% conc. H.sub.2SO.sub.4
solution in ethanol, followed by heating, or 1% aqueous KMnO.sub.4
followed by heating.
Column chromatography was performed with flash silica gel
(0.04-0.063 mm, Merck) or with ready-to-use Chromabon RS 40 flash
chromatography columns (Macherey-Nagel). All compounds were
characterized using .sup.1H and .sup.13C Nuclear Magnetic Resonance
(NMR) spectroscopy (Bruker Avance DPX-300 spectrometer, .sup.1H at
300.13 MHz and .sup.13C at 75.46 MHz). Assignments were made by
.sup.1H-.sup.1H COSY, DEPT and HSQC experiments. Chemical shifts
(.delta.) are given in parts per million (ppm) relatively to
tetramethylsilane or residual solvent peaks (CHCl.sub.3: .sup.1H:
7.26, .sup.13C: 77.0). Coupling constants J are given in Hertz
(Hz); peak multiplicity is reported as follows: s=singlet, bs=broad
singlet, d=doublet, t=triplet, m=multiplet.
High resolution electronspray ionization mass spectra (HR ESI-MS)
were performed by the CESAMO (Bordeaux, France) on a QSsat Elite
mass spectrometer (Applied Biosystems). The instrument is equipped
with an ESI source and spectra were recorded in negative mode. The
electrospray needle was maintained at 4500 V and operated at room
temperature. Samples were introduced by injection through a 10
.mu.L sample loop into a 200 .mu.L/min flow of methanol from the LC
pump.
The following abbreviations are used:
DCM dichloromethane
DMF dimethylformamide
DMSO dimethylsulfoxide
TBAI tetrabutylammonium iodide
THF tetra hydrofuran
The examples below, entitled "Preparation" describe the preparation
of synthesis intermediates used for preparing the compounds of
formula (I). The preparation of the compounds of formula (I) and
their applications are then described as "Examples". When present,
the number that accompanies the title compound in the "Preparation"
or in the "Example" refers to that shown in the schemes of FIGS. 1,
2 and 3.
FIG. 1 shows the synthetic scheme used in the Preparations and
Examples for compounds of formula (I) in which X is oxygen.
FIG. 2 shows the synthetic scheme used in the Preparations and
Examples for compounds of formula (I) in which X is
NH--C(O)--NH--.
FIG. 3 shows the synthetic scheme used in the Preparations and
Examples for compounds of formula (I) in which X is
--NH--C(O)--.
FIG. 4 shows the frequency sweep results for hydrogels obtained
from compounds 5 and 6.
FIG. 5 shows the step-strain measurement of the hydrogel obtained
from compound 6.
FIG. 6 shows the determination of the cytotoxicity of compound 6 by
MTT test on human mesenchymal stem cells.
FIG. 7 shows the cytocompatibility of compound 6 by monitoring
alamar blue metabolism in rat osteoblastic cells (A) or human stem
cells ASCs (B) grown in compound 6-based gels.
FIG. 8 shows the cytotoxycity of compound GNL of G. Godeau et al.,
Chem. Comm., 2009, 34, 5127-5130_by MTT test in human hepatic
carcinoma cells HuH-7 (comparative example).
PREPARATION 1
1,12-Dipropargyloxydecane (2)
To a cooled (0.degree. C.) solution of compound 1 (commercially
available) (5 g, 24.7 mmol) in anhydrous DMF (60 mL) was added
sodium hydride in small portions (55% in mineral oil, 6.5 g, 148.2
mmol) under stirring. Propargyl bromide (80% w/w in toluene, 11 g,
74.1 mmol) was added followed by TBAI (0.91 g, 2.4 mmol), and
stirring was continued for 15 hours at room temperature. The
reaction was quenched with methanol (10 mL) and stirred a further
30 min. After addition of DCM (200 mL) the solution was washed with
water (3.times.50 mL) then brine (50 mL), the organic layer dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. Product
2 was isolated after purification on silica gel (hexane/ethyl
acetate 100/0 then 97/3) as a colorless liquid. Yield: 4.48 g
(65%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.22-1.43 (m, 16H,
CH.sub.2), 1.52-1.66 (m, 4H, CH.sub.2CH.sub.2O), 2.43 (t, J=2.4 Hz,
2H, CH propargyl), 3.52 (t, J=6.6 Hz, 4H, CH.sub.2CH.sub.2O), 4.15
(d, J=2.4 Hz, 4H, CH.sub.2 propargyl).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 26.08, 29.42, 29.50,
29.55 (CH.sub.2, CH.sub.2CH.sub.2O), 57.97 (CH.sub.2 propargyl),
70.31 (CH.sub.2O), 74.01 (CH propargyl).
PREPARATION 2
12-Propargyloxydodecan-1-ol (7)
To a cooled (0.degree. C.) solution of compound 1 (commercially
available) (4 g, 20 mmol) in anhydrous DMF (100 mL) was added
sodium hydride in small portions (55% in mineral oil, 2.5 g, 109
mmol) under stirring. Propargyl bromide (80% w/w in toluene, 9 g,
60 mmol) was added dropwise, and stirring was continued for 12
hours at low temperature. The reaction mixture was concentrated
under reduced pressure. The resulting mixture was poured into water
and extracted with DCM. The extracts were combined, washed with
water (3.times.50 mL) then brine (50 mL). The organic layer was
dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure.
Product 7 was isolated after purification on silica gel
(hexane/ethyl acetate 6/4) as a brown solid. Yield: 2.24 g
(46%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.19-1.30 (m, 16H,
CH.sub.2), 1.46-1.59 (m, 4H, CH.sub.2CH.sub.2O), 2.37 (large s, 1H,
OH), 2.40 (t, J=2.4 Hz, 1H, CH propargyl), 3.46 (t, J=6.6 Hz, 2H,
CH.sub.2O), 3.56 (t, J=6.7 Hz, 2H, CH.sub.2O), 4.08 (d, J=2.4 Hz,
CH.sub.2 propargyl).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 25.74, 26.03
(CH.sub.2), 29.39, 29.43, 29.53, 29.58
(CH.sub.2+CH.sub.2CH.sub.2O), 32.70 (CH.sub.2CH.sub.2O), 57.92
(CH.sub.2 propargyl), 62.72, 70.22 (CH.sub.2O), 74.13 (CH
propargyl).
PREPARATION 3
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazol-1-yl)]-5'-deoxy
thymidine (3)
To a solution of 7 obtained in Preparation 2 (1.50 g, 5.39 mmol)
and 5'-azido-5'-deoxythymidine (3.02 g, 11.30 mmol) in 140 mL of
THF/H.sub.2O (1:1) was added copper sulfate (172 mg, 1.07 mmol)
followed by sodium ascorbate (427 mg, 2.15 mmol). The mixture was
stirred at 60.degree. C. for 20 hours. After cooling to room
temperature, the solvents were removed under reduced pressure. The
resulting solid was washed with water until the washings were
colorless, and then with ethanol. After drying under high vacuum,
the resulting white solid was used in the next step without further
purification. Yield: 4.00 g (91%).
.sup.1H NMR (300 MHz, in DMSO-d.sub.6) .delta. 1.22 (s, 16H,
8CH.sub.2), 1.41-1.52 (m, 4H, CH.sub.2CH.sub.2O), 1.79 (s, 6H,
CH.sub.3 thymine), 2.08-2.15 (m, 4H, H-2'), 3.35-3.41 (m, 4H,
CH.sub.2O), 4.06-4.10 (m, 2H, H-4'), 4.24-4.29 (m, 2H, H-3'), 4.27
(s, 4H, OCH.sub.2-triazole), 4.57-4.74 (m, 4H, H-5'), 5.52 large s,
2H, OH thymine), 6.16 (t, J=7.5 Hz, 2H, H-1'), 7.33 (s, 2H, H-6
thymine), 8.06 (s, 2H, CH triazole), 11.34 (s, 2H, NH thymine).
.sup.13C NMR (75 MHz, in DMSO-d.sub.6) .delta. 12.53 (CH.sub.3
thymine), 6.11, 29.33, 29.48, 29.55 (CH.sub.2), 38.31 (C-2'), 51.51
(C-5'), 63.68 (CH.sub.2O-triazole), 70.01 (CH.sub.2O), 71.16
(C-3'), 84.40 (C1' and C-4'), 125.00 (CH triazole), 136.45 (C-6
thymine).
PREPARATION 4
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazol-1-yl)]-5'-deoxy-N-3--
propargyl thymidine (4)
To as solution of compound 3 obtained in Preparation 3 (3.50 g,
4.30 mmol) in anhydrous DMF (120 mL) was added sodium carbonate
(1.7 g, 12.92 mmol) followed by propargyl bromide (80% w/w in
toluene, 1.92 g, 12.92 mmol) and TBAI (0.16 g, 043 mmol). The
mixture was stirred at room temperature for 20 hours, then poured
into DCM (200 mL) and washed with water (3.times.50 mL) and brine
(50 mL). The organic extract was dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The product was purified by
column chromatography on silica gel eluting with DCM/MeOH (100:0 to
96:4) and obtained as a white solid foam. Yield: 3.07 g (77%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.27 (s, 16H, 8
CH.sub.2), 1.53-1.60 (m, 4H, 2 CH.sub.2CH.sub.2O), 1.94 (s, 6H, 2
CH.sub.3 thymine), 2.22 (t, 2H, J=2.3 Hz, propargylic CH),
2.31-2.41 (m, 4H, 2H-2'), 3.52 (t, 4H, J=6.5 Hz, 2 CH.sub.2O),
4.23-4.27 (m, 2H, 2H-4'), 4.53-4.59 (m, 2H, 2H-3'), 4.61 (s, 4H, 2
CH.sub.2O triazole), 4.71 (s, 4H, 2 propargylic CH.sub.2),
4.70-4.77 (m, 4H, 2H-5'), 6.23 (t, 2H, J=6.6 Hz, 2H-1'), 6.81 (s,
2H, 2H-6 thymine), 7.68 (s, 2H, 2H triazole).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 13.16 (CH.sub.3
thymine), 29.22, 29.31, 29.34, 29.47 (CH.sub.2), 30.50 (propargyl
CH.sub.2), 38.61 (C-2'), 51.11 (C-5'), 63.93 (OCH.sub.2 triazole),
71.14 (CH.sub.2O), 71.23 (C-3'), 77.25 (propargyl CH), 83.79
(C-4'), 86.85 (C-1'), 124.60 (CH triazole), 134.72 (C-6
thymine).
PREPARATION 5
5'-[4-((12-Hydroxydodecanyloxy)methyl)1H-1,2,3-triazol-1-yl]-5'-deoxy
thymidine (8)
To a degassed solution of compound 7 obtained in Preparation 1
(2.23 g, 9.30 mmol) in 80 mL THF/H.sub.2O (1:1) was added
5'-azido-5'-deoxy thymidine (2.48 g, 9.30 mmol), copper sulfate
(0.15 g, 0.93 mmol) and sodium ascorbate (0.37 g, 1.86 mmol). The
mixture was stirred at 60.degree. C. for 3 hours. After removal of
THF in vacuo, the aqueous mixture was filtered, the resulting
yellow solid was washed with water, then purified by column
chromatography on silica gel eluting with DCM/MeOH (9:1) to elute
the product as a white solid.
Yield: 3.88 g (82%).
.sup.1H NMR (300 MHz, in MeOD @ 313K) .delta. 1.30-1.39 (m, 16H,
CH.sub.2), 1.52-1.60 (m, 4H, CH.sub.2CH.sub.2O), 1.89 (d, J=1.2 Hz,
3H, CH.sub.3 thymine), 2.23-2.29 (m, 2H, H-2'), 3.49-3.57 (m, 4H
chain CH.sub.2O+CH.sub.2OH), 4.16-4.21 (m, 1H, H-4'), 4.39-4.43 (m,
1H, H-3'), 4.72-4.81 (m, 2H, H-5'), 6.20 (t, J=6.7 Hz, H-1'), 7.20
(d, 1H, H-6 thymine), 7.95 (s, 1H, CH triazole).
.sup.13C NMR (75 MHz, in MeOD @ 313K) .delta. 10.95 (CH.sub.3
thymine), 25.51, 25.74, 29.06, 29.13, 29.22, 29.27, 32.25
(CH.sub.2), 38.26 (C-2'), 51.09 (C-5'), 61.64 (CH.sub.2OH or
CH.sub.2CH.sub.2O), 63.27 (CH.sub.2O triazole), 70.38
(CH.sub.2CH.sub.2O or CH.sub.2OH), 70.98 (C-3'), 84.06 (C-4'),
85.46 (C-1'), 110.10 (C-5 thymine), 124.61 (CH triazole), 136.55
(C-6 thymine), 150.30 & 164.31 (C.dbd.O).
HRMS: (M+Na) 530.2943 (calculated 530.2949)
PREPARATION 6
5'-[4-((12-Hydroxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5'deoxy-N-3--
propargylthymidine (9)
To a solution of compound 8 obtained in Preparation 5 (1.27 g, 25.0
mmol) in anhydrous DMF (60 mL) was added potassium carbonate (0.69
g, 50.0 mmol) and TBAI (0.09 g, 2.5 mmol), followed by propargyl
bromide (80% w/w in toluene, 1.19 g, 50.0 mmol). The mixture was
stirred at room temperature for 24 hours, then filtered, and the
filtrate was concentrated under reduced pressure. The resulting
yellow oil was poured into water (50 mL) and extracted with DCM
(3.times.50 mL). The combined organic extracts were washed with
water (50 mL), then brine (50 mL), dried (Na.sub.2SO.sub.4), and
the solvent removed in vacuo. The crude product was purified by
column chromatography on silica gel eluting with DCM/MeOH (95:5) to
elute the title compound as a viscous yellow oil which crystalizes
on standing (white solid). Yield: 1.20 g (87%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.26-1.34 (m, 16H,
CH.sub.2), 1.51-1.62 (m, 4H, CH.sub.2CH.sub.2OH+CH.sub.2CH.sub.2O),
1.93 (d, J=1.1 Hz, 3H, CH.sub.3 thymine), 2.20 (t, J=2.4 Hz, 1H,
propargylic CH), 2.22-2.29 (m, 1H, H-2'a), 2.35-2.44 (m, 1H,
H-2'b), 3.48-3.56 (m, 2H CH.sub.2O), 3.65 (t, J=6.6 Hz, 2H,
CH.sub.2OH), 4.18-4.23 (m, 1H, H-4'), 4.45-4.51 (m, 1H, H-3'), 4.59
(s, 2H triazole CH.sub.2O), 4.70-4.74 (m, 4H, H-5'+propargylic
CH.sub.2), 6.25 (t, J=6.6 Hz, H-1'), 6.74 (d, 1H, H-6 thymine),
7.68 (s, 1H, CH triazole).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 12.93 (CH.sub.3
thymine), 25.68, 25.91, 29.30, 29.33, 29.41, 29.47
(CH.sub.2+CH.sub.2CH.sub.2OH or CH.sub.2CH.sub.2O), 30.36
(propargylic CH.sub.2), 32.55 (CH.sub.2CH.sub.2O or
CH.sub.2CH.sub.2OH), 38.58 (C-2'), 51.00 (C-5'), 62.49
(CH.sub.2OH), 63.85 (CH.sub.2 triazole), 70.65 (C-3'), 70.85
(propargylic CH), 70.99 (triazole CH.sub.2O), 83.77 (C-4'), 86.30
(C-1'), 124.48 (CH triazole), 134.83 (C-6 thymine).
HRMS: (M+Na) 568.3098 (calculated 568.3105)
PREPARATION 7
5'-[4-((12-Propargyloxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5'-deox-
y-N-3-propargylthymidine (10)
To a cold (0.degree. C.) solution of compound 8 obtained in
Preparation 5 (2 g, 3.66 mmol) in anhydrous DMF (100 mL) was added
sodium hydride in one portion (55% in mineral oil, 0.90 g, 22
mmol). After 15 min, TBAI (0.135 g, 0.36 mmol) was added followed
by propargyl bromide in small portions (80% w/w in toluene, 1.63 g,
11 mmol), and stirring continued for 5 hours at 0.degree. then at
room temperature overnight. The reaction was quenched with water
(10 mL) with stirring and cooling on ice, and the solution was
concentrated under reduced pressure. The brown residue was
dissolved in water (200 mL) and extracted with DCM (3.times.100 mL)
The combined organic extracts were washed with brine (3.times.100
mL) and aqueous 10% KCl solution (100 mL), dried (Na.sub.2SO.sub.4)
and the solvent removed in vacuo. The crude product was purified by
chromatography on silica gel eluting with ethyl acetate and
obtained as a yellow oil. Yield: 0.87 g (40%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.23-1.27 (m, 16H,
CH.sub.2), 1.48-1.56 (m, 4H, CH.sub.2CH.sub.2O), 1.89 (s, 3H,
CH.sub.3 thymine), 2.08-2.17 (m, 1H, H-2'a), 2.18 (m, 1H thymine
propargylic CH), 2.35-2.43 (m, 1H, H-2'b), 2.51 (m, 1H, chain
propargylic CH), 3.48 (t, J=6.7 Hz, 2H CH.sub.2OCH.sub.2-triazole),
3.59 (t, J=6.6 Hz, 2H, chain CH.sub.2O-propargyl), 4.17-4.20 (m,
2H, propargylic CH.sub.2O), 4.24-4.30 (m, 1H, H-4'), 4.35-4.40 (m,
1H, H-3'), 4.57 (s, 2H CH.sub.2 triazole), 4.57-4.70 (m, 4H,
H-5'+propargylic CH.sub.2N), 6.14 (t, J=6.9 Hz, H-1'), 6.73 (s, 1H,
H-6 thymine), 7.62 (s, 1H, CH triazole).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 13.06 (CH.sub.3
thymine), 25.73, 26.03, 29.49, 29.54 (CH.sub.2), 29.39
(CH.sub.2CH.sub.2O), 30.42 (thymine propargylic CH.sub.2), 32.76
(CH.sub.2CH.sub.2O propargyl), 36.23 (C-2'), 50.92 (C-5'), 57.41
(propargylic CH.sub.2O), 62.84 (chain CH.sub.2O-propargyl), 64.16
(CH.sub.2 triazole), 70.80 (thymine propargylic CH), 71.08 (chain
CH.sub.2OCH.sub.2 triazole), 75.70 (chain propargylic CH), 78.26
(C-3'), 81.76 (C-4'), 86.69 (C-1'), 124.18 (CH triazole), 134.30
(C-6 thymine).
HRMS: (M+Na) 606.3258 (calculated 606.3262)
PREPARATION 8
1,1'-(dodecane-1,12-diyl)bis(3-[propargyl]urea) (12)
To a solution of 1,12-diisocyanatododecane (0.53 mL, 2 mmol) in
anhydrous DCM (20 mL) was added slowly propargylamine (0.30 mL, 4.8
mmol). The mixture was stirred for 16 hours at room temperature.
The precipitate was filtered and washed abundantly with DCM to
remove the excess of propargylamine. After drying, the product was
obtained as a white solid.
Yield: (99%).
.sup.1H NMR (300 MHz, in DMSO-d.sub.6 @ 350 K) .delta. (ppm)
1.50-1.63 (m, 20H), 3.15 (t, J=2.6 Hz, 2H), 3.20-3.26 (q, 4H), 4.03
(m, J=2.6 Hz, 4H), 6.08-6.25 (large s, 4H).
.sup.13C NMR (75 MHz, DMSO-d.sub.6, 350 K) .delta. (ppm) 26.82,
27.91, 28.05, 29.26, 29.32, 29.69, 30.34, 31.0, 31.09, 72.58,
83.05, 158.02.
PREPARATION 9
N3-propargylthymidine (13)
To a solution of thymidine (3 g, 12.39 mmol) in anhydrous DMF (20
mL) was added potassium carbonate (2.58 g, 18.58 mmol), propargyl
bromide (80% w/w in toluene, 2.07 mL, 18.58 mmol). The reaction was
stirred for 2 days at room temperature. After removal of DMF in
vacuo, the residue was dissolved in ethyl acetate (100 mL) and then
washed with water (3.times.30 mL). The organic extract was dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
resulting oil was used in the next step without further
purification. Yield: 3 g (86%).
.sup.1H NMR (300 MHz, in DMSO d.sub.6) .delta. 1.85 (s, 3H),
2.10-2.18 (m, 2H), 3.10 (s, 1H), 3.59 (m, 2H), 3.79 (s, 1H), 4.25
(m, 1H), 4.53 (s, 2H), 5.04 (t, J=6 Hz, 1H), 5.27 (s, 1H), 6.21 (t,
J=6 Hz, 1H), 7.82 (s, 1H).
.sup.13C NMR (75 MHz, in DMSO d.sub.6) .delta. 13.0, 30.3, 40.3,
62.0, 70.9, 71.1, 78.2, 86; 0, 87.1, 116.9, 135.2, 150; 2,
162.7.
HRMS: (M+H) 281, 1135 (calculated 281.1137)
PREPARATION 10
Compound (14)
##STR00001##
5'-deoxy-N3-(1-(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3--
triazol-4-yl) thymidine (14)
To a solution of 13 obtained in Preparation 9 (2.80 g, 10 mmol) and
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranosyl azide (3.73 g, 10
mmol) in 80 mL of tert-butanol/H.sub.2O (1:1) was added copper
sulfate (159 mg, 1 mmol) and sodium ascorbate (396 mg, 2 mmol). The
mixture was stirred at 60.degree. C. for 20 hours. The solvent was
removed under reduced pressure and the residual solid was dissolved
in ethyl acetate (150 mL) and then washed with water (3.times.50
mL). The organic extract was dried (Na.sub.2SO.sub.4) and
concentrated under reduced pressure. The crude product was purified
by chromatography on silica gel eluting with ethyl acetate and
obtained as a white solid. Yield: 5.33 g (82%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.84-2.08 (m, 15H),
2.35 (m, 2H), 3.82-3.92 (m, 2H), 4.00 (m, 2H), 4.11-4.15 (dd, 1H),
4.25-4.31 (dd, 1H), 4.58 (m, 1H), 5.19-5.31 (m, 3H), 5.36-5.48 (m,
2H), 5.85 (d, J=8.8 Hz, 1H), 6.23 (t, J=6.4 Hz, 1H), 7.52 (s, 1H),
7.88 (s, 1H).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 12.47, 19.93, 20.32,
20.47, 35.18, 39.60, 61.00, 61.22, 67.03, 69.58, 70.15, 72.03,
74.14, 84.72, 85.45, 86.62, 99.37, 109.15, 122.30, 134.58, 142.94,
149.97, 162.53, 168.23, 168.77, 169.22, 169.99.
PREPARATION 11
Compound (15)
##STR00002##
5'-Azido-N3-(1-(2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3--
triazol-4-yl) thymidine (15)
To a solution of 14 obtained in Preparation 10 (1 g, 1.53 mmol) in
anhydrous DMF (6 mL) was successively added PPh.sub.3 (0.48 g, 1.84
mmol), NaN.sub.3 (0.50 g, 7.65 mmol) and CBr.sub.4 (0.61 g, 1.84
mmol). After stirring for 24 hours, the reaction was quenched with
methanol (1.5 mL) and stirred a further 30 min. The solvent was
removed under reduced pressure and the product was isolated after
purification on silica gel (DCM/MeOH 100/0 then 97/3) as a white
solid. Yield: 1.31 g (77%).
.sup.1H NMR (300 MHz, in CDCl.sub.3) .delta. 1.72-1.97 (m, 15H),
2.10-2.17 (m, 1H), 2.30-2.33 (m, 1H), 3.49-3.65 (m, 2H), 4.00-4.22
(m, 3H), 4.25-4.31 (dd, 1H), 4.38 (m, 1H), 5.05-5.22 (m, 3H),
5.35-5.45 (m, 2H), 5.85 (d, J=8.8 Hz, 1H), 6.23 (t, J=6.4 Hz, 1H),
7.33 (s, 1H), 7.85 (s, 1H).
.sup.13C NMR (75 MHz, in CDCl.sub.3) .delta. 12.47, 19.98-20.53,
35.76, 39.97, 52.09, 61.50, 67.56, 70.06, 71.07, 72.58, 74.72,
84.44, 85.32, 110.17, 122.68, 134.04, 143.49, 150.44, 162.87,
168.68, 170.50.
PREPARATION 12
1,1'-(tetradecane-1,14-diyl)bis(3-[propargyl]amide) (18)
Thionyl chloride (9.12 mmol, 2.1 eq) and triethylamine (13.02 mmol,
3 eq) was added dropwise to a suspension of hexadecanedioic acid
(4.34 mmol) in dry DCM. The mixture was vigorously stirred for 1
hour at room temperature. Then, propargylamine (9.55 mmol, 2.2 eq)
was added slowly. After stirring for 16 hours, the mixture was
filtrated and the crude product was washed abundantly with water
and ethanol. Yield: 36%.
.sup.1H NMR (300 MHz, DMSO-d.sub.6, 340 K) .delta. 1.25-1.49 (m,
24H), 2.08 (t, 4H,), 2.93 (t, 2H), 3.83 (m, 4H), 7.99 (large s,
4H).
.sup.13C NMR (75 MHz, DMSO-d.sub.6, 340 K) .delta. 24.6, 27.3,
28.2, 28.3, 28.4, 28.5, 34.7, 71.9, 81.0, 171.5.
Example 1
5'-[4-(12-hydroxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5'-deoxy-N3-(-
1-((.beta.-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl
thymidine (5)
To a degassed solution of compound 9 obtained in Preparation 6
(1.182 g, 2.0 mmol) and 1-azido-.beta.-D-glucopyranose (0.534 g,
2.0 mmol) in 20 mL of THF/H.sub.2O (1:1) was added copper sulfate
(32 mg, 0.2 mmol) followed by sodium ascorbate (80 mg, 0.4 mmol).
The mixture was stirred at 60.degree. C. for 20 hours. After
cooling to room temperature, the solvents were removed under
reduced pressure. The resulting green solid was dissolved in
methanol and filtered through celite, the resulting solution was
concentrated under reduced pressure and applied to a column of
silica gel. The product was eluted with DCM/MeOH (9:1 to 8:2).
Yield: 0.84 g (55%).
.sup.1H NMR (300 MHz, in MeOD) .delta. 1.28-1.35 (s, 18H,
CH.sub.2), 1.51-1.60 (m, 4H, CH.sub.2CH.sub.2O), 1.94 (s, 3H,
CH.sub.3 thymine), 2.28-2.32 (dd, J=6.0 Hz, 2H, H-2'), 3.45-3.60
(m, 5H, H-3, H-4, H-5, CH.sub.2O), 3.66-3.72 (dd, J=12.0, 5.2 Hz,
2H, H-6a), 3.84-3.91 (m, 2H, H-2, H-6b), 4.19-4.21 (m, 1H, H-4'),
4.42-4.45 (m, 1H, H-3'), 4.56 (s, 2H, CH.sub.2 triazole), 4.75-4.78
(m, 2H, H-5'), 5.22 (s, 2H, CH.sub.2--N triazole), 5.58 (d, J=9.2
Hz, 1H, H-1), 6.24 (dd, J=6.0 Hz, 1H, H-1'), 7.31 (s, 1H, H-6
thymine), 8.00 (s, 1H, H triazole), 8.11 (s, 1H, H triazole).
.sup.13C NMR (75 MHz, in MeOD) .delta. 11.80 (CH.sub.3 thymine),
25.56, 25.80, 29.23, 29.26, 32.27 (CH.sub.2 and CH.sub.2CH.sub.2O),
35.62 (CH.sub.2N triazole), 38.21 (C-2'), 51.21 (C-5'), 60.92
(C-6), 61.60 (CH.sub.2O), 63.14 (OCH.sub.2 triazole), 70.33
(CH.sub.2O), 69.41, 76.93, 79.67 (C-3 or C-4 or C-5), 71.00 (C-3'),
72.50 (C-2), 84.27 (C-4'), 86.53 (C-1'), 88.15 (C-1), 109.82 (C-5
thymine), 123.03, 124.82 (CH triazole), 135.47 (C-6 thymine),
143.12, 144.72 (C quat. triazole), 150.59, 163.37 (C.dbd.O
thymine).
HRMS: (M+Na) 773.3795 (calculated 773.3804)
Example 2
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((.be-
ta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine (6)
To a solution of 4 obtained in preparation 4 (200 mg, 0.225 mmol)
and 1-azido-.beta.-D-glucopyranose (111 mg, 0.540 mmol) in 12 mL of
THF/H.sub.2O (1:1) was added copper sulfate (14 mg, 0.09 mmol)
followed by sodium ascorbate (53 mg, 0.27 mmol). The mixture was
irradiated under stirring in a microwave reactor (open vessel
equipped with a reflux condenser) at 200 W and 75.degree. C. for 5
min. The crude resulting mixture was concentrated under reduced
pressure and purified by column chromatography on silica gel
eluting with DCM/MeOH (8:2 to 7:3) to give 5 then 6. Yields: 5, 16
mg (9.5%); 6, 177 mg (58%).
.sup.1H NMR (300 MHz, in MeOD) .delta. 1.28-1.35 (s, 16H,
CH.sub.2), 1.52-1.61 (m, 4H, CH.sub.2CH.sub.2O), 1.92 (s, 6H,
CH.sub.3 thymine), 2.27-2.31 (dd, J=6.0 Hz, 4H, H-2'), 3.48-3.60
(m, 10H, H-3, H-4, H-5, CH.sub.2O), 3.67-3.73 (dd, J=12.0, 5.2 Hz,
2H, H-6a), 3.85-3.93 (m, 4H, H-2, H-6b), 4.17-4.22 (m, 2H, H-4'),
4.41-4.46 (m, 2H, H-3'), 4.56 (s, 4H, triazole CH.sub.2O),
4.74-4.77 (m, 4H, H-5'), 5.21 (s, 4H, triazole CH.sub.2N), 5.59 (d,
J=9.2 Hz, 2H, H-1), 6.23 (dd, J=6.0 Hz, 2H, H-1'), 7.28 (s, 2H, H-6
thymine), 7.99 (s, 2H, H triazole), 8.13 (s, 2H, H triazole).
.sup.13C NMR (75 MHz, in MeOD) .delta. 11.80 (CH.sub.3 thymine),
25.76 (CH.sub.2), 29.23 (CH.sub.2CH.sub.2O), 35.67 (CH.sub.2N
triazole), 38.21 (C-2'), 48.45 (C-5'), 60.95 (C-6), 63.16
(OCH.sub.2 triazole), 69.42, 76.98, 79.69 (C-3, C-4, C-5), 70.32
(CH.sub.2O), 70.98 (C-3'), 72.51 (C-2), 84.23 (C-4'), 86.63 (C-1'),
88.17 (C-1), 122.91, 124.79 (CH triazole), 135.44 (C-6
thymine).
HRMS: (M+Na) 1321.5802 (calculated 1321.5783)
Example 3
5'-[4-((.beta.-D-Glucopyranosyloxy)dodecanyloxy)methyl)-1H-1,2,3-triazol-1-
-yl]-5'-deoxy-N-3-[1-((.beta.-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)met-
hyl]thymidine (11)
To a degassed solution of compound 10 obtained in Preparation 7
(0.87 g, 1.49 mmol) in 24 mL THF/H.sub.2O (6:4) was added
1-azido-.beta.-D-glucopyranose (0.62 g, 3.00 mmol), copper sulfate
(120.00 mg, 0.60 mmol) and sodium ascorbate (47.80 mg, 0.30 mmol).
The mixture was stirred at 60.degree. C. for 3 hours. After removal
of the solvents in vacuo, the resulting green solid was dissolved
in methanol and filtered through celite, the resulting solution was
concentrated under reduced pressure and applied to a column of
silica gel. The product was eluted with DCM/MeOH (8:2). Yield: 0.81
g (54%).
.sup.1H NMR (300 MHz, in MeOD) .delta. 1.30-1.33 (m, 16H,
CH.sub.2), 1.50-1.62 (m, 4H, chain-CH.sub.2CH.sub.2O), 1.93 (d,
J=1.0 Hz, 3H, CH.sub.3 thymine), 2.26-2.47 (m, 1H, H-2'), 3.49-3.60
(m, 10H, 2 chain-CH.sub.2O, H-3A, H-3B, H-4A, H-4B, H-5A, H-5B),
3.67-3.76 (2 dd, 2H, J=11.6, 5.2 Hz, H-6A, a and H-6B, a),
3.85-3.97 (m, 4H, H-6A, b, H-6B, a and H-2A, H-2B), 4.33-4.37 (m,
2H, H-3' and H-4'), 4.56 (s, 2H, triazole-CH.sub.2O), 4.68-4.70 (d,
J=4.7 Hz, 2H, triazole-CH.sub.2O), 4.74-4.76 (m, 2H, H-5'), 5.22
(s, 2H, triazole-CH.sub.2N), 5.58 (d, J=9.2 Hz, 1H, H-1A or H-1B),
5.65 (d, J=9.2 Hz, 1H, H-1B or H-1A), 6.19 (t, J=7.0 Hz, H-1'),
7.30 (d, 1H, H-6 thymine), 7.98, 8.13 and 8.25 (3s, 1H, CH
triazole).
.sup.13C NMR (75 MHz, in MeOD) .delta. 11.75 (CH.sub.3 thymine),
25.55, 25.80, 29.18, 29.22, 29.26, 29.33 (CH.sub.2 and
CH.sub.2CH.sub.2O), 32.26 (CH.sub.2CH.sub.2O), 32.50
(triazole-CH.sub.2-thymine), 51.07 (C-5'), 60.94 (C-6A, C-6B),
61.58 (chain-CH.sub.2O), 61.93 and 63.11 (triazole-CH.sub.2O),
69.40, 69.44 (C-3, C-4 or C-5 A and B), 70.36 (chain-CH.sub.2O),
72.49, 72.61 (C-2A, C-2B), 76.98, 77.01 (C-3, C-4 or C-5 A and B),
78.49, 82.12 (C-3', C-4'), 79.69, 79.74 (C-3, C-4 or C-5 A and B),
87.04 (C-1'), 88.17, 88.25 (C-1A, C-1B), 122.93, 123.18, 124.79 (CH
triazole), 135.53 (C-6 thymine).
HRMS: (M+Na) 1016.4648 (calculated 1016.4859).
Example 4
1,12-bis-dodecanyl-5'-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-5'-deoxy-3'-O--
1-((.beta.-D-glucopyranoside)-1-H-1,2,3-triazol-4-yl)methyl
thymidine
##STR00003##
To a degassed solution of 7 obtained in preparation 1 (139.0 mg,
0.5 mmol) in 30 mL THF/H.sub.2O (2:1) was added
5'-azido-5'-deoxy-3'-O-1-((.beta.-D-glucopyranoside
tetraacetate)-1H-1,2,3-triazol-4-yl)methyl thymidine [L. Latxague,
A. Patwa, E. Amigues, P. Barthelemy, Molecules 18 (2013) 12241-63],
copper sulfate (15.9 mg, 0.1 mmol) and sodium ascorbate (39.6 mg,
0.2 mmol). The mixture was stirred at 65.degree. C. for 6 hours
then overnight at rt. After removal of the solvents in vacuo, the
remaining crude material was applied to a column of silica gel. The
product was eluted with DCM/MeOH (95:5) then subjected to a Zemplen
deacetylation. Yield: 301 mg (46%).
.sup.1H NMR (300 MHz, in MeOD @ 323K) .delta. 1.30 (s, 16H,
CH.sub.2), 1.55-1.63 (m, 4H, CH.sub.2CH.sub.2O), 1.91 (s, 6H,
CH.sub.3 thymine), 2.22-2.31 (m, 2H, H-2'a), 2.39-2.47 (m, 2H,
H-2'b), 3.39 (t, J=6.5 Hz, 4H, CH.sub.2CH.sub.2O), 3.54-3.66 (m,
6H, H-3, 4, 5), 3.74-3.81 (m, 2H, H-6a), 3.91-4.00 (m, 4H, H-2,
6b), 4.34-4.39 (m, 4H, H-3', 4'), 4.60 (s, 4H, OCH.sub.2 triazole),
4.71-4.76 (m, 8H, H-5' and OCH.sub.2 triazole), 5.66 (d, J=9.2 Hz,
2H, H-1), 6.18 (dd, J=6.7 Hz, 2H, H-1'), 7.22 (s, 2H, H-6 thymine),
7.96 (s, 2H, H triazole), 8.21 (s, 2H, H triazole).
.sup.13C NMR (75 MHz, in MeOD @ 323K) .delta. 9.75 (CH.sub.3
thymine), 24.49 (CH.sub.2), 27.76, 27.89, 27.97 (CH.sub.2 and
CH.sub.2CH.sub.2O), 34.39 (C-2'), 49.87 (C-5'), 59.91 (C-6), 60.97
and 62.07 (CH.sub.2 triazole), 68.47, 75.95, 78.55 (C, 3, 4, 5),
69.20 (CH.sub.2CH.sub.2O), 71.49 (C-2), 77.42, 80.77 (C-3', 4'),
84.60 (C-1'), 87.08 (C-1), 109.44 (C-5 thymine), 121.88, 123.43 (CH
triazole), 135.32 (C-6 thymine), 142.91, 143.87 (C-4 triazole),
149.51, 163.46 (C.dbd.O thymine).
Example 5
1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5'-[4-(oxymethy-
l)-1H-1,2,3-triazol-1-yl]-N-3-[1-((.beta.-D-glucopyranoside)-1H-1,2,3-tria-
zol-4-yl)methyl]-5'-deoxythymidine
##STR00004##
To a degassed solution of
1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5'-[4-(oxymeth-
yl)-1H-1,2,3-triazol-1-yl]-N-3-propargyl-5'-deoxythymidine obtained
from commercial
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-1,10-decanediol
subjected to a similar set of reactions as shown in FIG. 1, namely
propargylation/azidothymidine coupling/nucleobase propargylation)
(782.00 mg, 0.68 mmol) in 15 mL THF/H.sub.2O (2:1) was added
1-azido-1-deoxyglucopyranose, copper sulfate (26.90 mg, 0.14 mmol)
and sodium ascorbate (43.40 mg, 0.27 mmol). The mixture was stirred
at 60.degree. C. overnight. After removal of the solvents in vacuo,
the remaining crude material was washed with water and the filtrate
was concentrated under reduced pressure. The oily residue was
applied to a column of silica gel. The product was eluted with
DCM/MeOH (8:2 then 7:3). Yield: 514.00 mg (48%).
.sup.1H NMR (300 MHz, in MeOD) d 1.91 (d, J=1.1 Hz, 6H, CH.sub.3
thymine), 2.28-2.32 (t, J=6.2 Hz, 4H, H-2'), 3.47-3.58 (m, 6H,
H-2,3,5), 3.66-3.71 (dd, J=5.1, 12.1 Hz, 2H, H-6a), 3.83-3.90 (m,
4H, H-4, 6b), 4.09-4.18 (t, J=14.1 Hz, 4H, CH.sub.2CF.sub.2),
4.17-4.22 (m, 2H, H-4'), 4.40-4.45 (dd, J=5.2 Hz 2H, H-3'),
4.74-4.79 (m, 8H, H-5' and OCH.sub.2 triazole), 5.20 (s, 4H,
OCH.sub.2 triazole), 6.56 (d, J=9.2 Hz, 2H, H-1), 6.20 (dd, J=6.7
Hz, 2H, H-1'), 7.29 (d, 2H, H-6 thymine), 8.05 (s, 2H, H triazole),
8.10 (s, 2H, H-triazole).
.sup.13C NMR (75 MHz, in MeOD) d 10.14 (CH.sub.3 thymine), 34.15
(OCH.sub.2 triazole), 36.72 (C-2'), 49.76 (C-5'), 59.47 (C-6),
63.11 (OCH.sub.2 triazole), 64.97 (t, .sup.2J.sub.C-F=26.0 Hz,
CH.sub.2CF.sub.2), 67.98, 75.54, 78.22 (C2, 3 or 5), 69.60 (C-3'),
71.05 (C-4), 82.80 (C-4'), 85.44 (C-1'), 86.72 (C-1), 108.26 (C-5
thymine), 121.36, 123.75 (CH triazole), 134.00 (C-6 thymine),
141.91 (C-4 triazole), 149.05 and 161.87 (C.dbd.O thymine).
Example 6
1,12-bis-dodecanyl-5'-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3,-
4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl-
]-5'-deoxythymidine (16)
To a solution of 15 obtained in Preparation 11 (1 g, 1.47 mmol) in
36 mL of tert-butanol/H.sub.2O (1:1) was added compound 12 obtained
in Preparation 8 (243 mg, 0.67 mmol), copper sulfate (33 mg, 0.13
mmol) and sodium ascorbate (53 mg, 0.27 mmol). The mixture was
stirred at 75.degree. C. for 20 hours. The solvent was removed
under reduced pressure and the residual solid was dissolved in DCM
(150 mL) and then washed with water (3.times.50 mL). The organic
extract was dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure. The crude product was purified by chromatography on
silica gel eluting with ethyl acetate/MeOH (10:0 to 90:10) and
obtained as a white solid. Yield: 0.90 g (78%).
.sup.1H NMR (300 MHz, in DMSO-d.sub.6) .delta. 1.23-1.32 (m, 20H,
chain CH.sub.2), 1.76-2.00 (m, 15H), 2.17 (m, 2H), 2.95 (q, 4H),
4.08 (m, 6H), 4.20-4.36 (m, 8H), 4.57-4.73 (m, 4H), 4.97-5.11 (q,
4H), 5.16 (t, 2H), 5.54 (m, 2H), 5.66 (t, 2H), 6.21 (t, 2H), 6.31
(d, 2H), 7.50 (s, 2H), 7.85 (s, 2H), 8.26 (s, 2H).
.sup.13C NMR (75 MHz, in DMSO-d.sub.6) .delta. 13.12, 20.32-20.96,
35.41, 36.36, 38.54, 51.53, 62.27, 67.97, 70.38, 71.18, 72.68,
72.85, 73.73, 84.20, 84.74, 85.72, 109.18, 123.07, 135.55, 143.81,
150.61, 158.32, 162.69, 168.68, 169.82, 170.03, 170.51.
Example 7
1,12-bis-dodecanyl-5'-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((.bet-
a.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine (17)
A solution of sodium methoxide (1M in methanol, 0.2 mL) was added
dropwise to a solution of compound 16 prepared in example 6 (0.49
g, 0.28 mmol) in 20 mL of methanol anhydrous. After heating for 1 h
minutes at 60.degree. C., amberlite IRC-50 was added to convert
from Na.sup.+ to H.sup.+ ions. After 20 minutes at the same
temperature, the resin was removed by filtration and washed with
methanol. The filtrate was concentrated and the product was
purified by column chromatography on silica gel eluting with
MeOH:CH.sub.2Cl.sub.2 (30:70 to 50:50) to afford as a white solid
(Yield: 79%, 0.31 g).
.sup.1H NMR (300 MHz, in MeOD) .delta. 1.26-1.41 (m, 20H), 1.89 (s,
3H), 2.24-2.29 (m, 4H), 3.03-3.07 (t, 4H), 3.40-3.54 (m, 6H),
3.62-3.68 (dd, 2H), 3.81-3.86 (m, 4H), 4.06-4.16 (m, 2H), (4.12 (s,
4H), 4.32-4.39 (m, 2H), 4.60-4.74 (m, 4H), 4.02 (s, 6, 160.89,
164.79.
Example 8
1,14-bis-tetradecanyl-5'-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-((-
2,3,4,6-tetra-O-acetyl-.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)me-
thyl]-5'-deoxy thymidine (19)
To a solution of compound 18 (0.24 g, 0.67 mmol) and compound 15 (1
g, 1.47 mmol, 2.2 eq.) in 36 mL of tert-Butanol/H2O (1:1) was added
sodium ascorbate (53 mg, 0.27 mmol, 0.4 eq.) followed by copper
sulfate pentahydrate (33 mg, 0.13 mmol, 0.2 eq.). The mixture was
stirred at 75.degree. C. for 24 hours.
After cooling to room temperature, the solvents were removed under
reduced pressure. The resulting solid was dissolved in DCM and the
solution was washed with water. The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated. The product was
purified by column chromatography on silica gel eluting with ethyl
acetate/MeOH (100:0 to 80:20) to afford as a white solid (Yield:
76%, 0.87 g).
.sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta. 1.21 (s, 20H),
1.43-1.49 (m, 4H), 1.76-2.03 (m, 24H), 1.87 (s, 6H), 2.04-2.09 (t,
J=7.5 Hz, 4H), 2.12-2.23 (m, 4H), 4.03-4.14 (m, 6H), 4.26-4.37 (m,
8H), 4.58-4.73 (m, 4H), 4.97-5.11 (m, 4H), 5.14-5.20 (t, J=9.8 Hz,
2H), 5.49-5.54 (t, J=9.6 Hz, 2H), 5.49-5.54 (d, 2H), 5.63-5.69 (t,
J=9.4 Hz, 2H), 6.20-6.24 (t, J=6.6 Hz, 2H), 6.28-6.32 (d, J=9.2 Hz,
2H), 7.49 (s, 2H), 7.90 (s, 2H), 8.25 (s, 2H), 8.25-8.29 (m,
2H).
.sup.13C NMR (75 MHz, DMSO-d.sub.6) .delta. 12.68, 20.33, 20.70,
20.84, 20.97, 25.65, 29.16, 29.26, 29.41, 29.53, 34.07, 35.21,
35.92, 38.12, 51.11, 61.83, 67.52, 70.00, 70.72, 72.22, 73.29,
83.74, 84.27, 85.24, 109.05, 122.62, 123.48, 135.11, 143.33,
145.14, 150.17, 162.23, 168.42, 169.36, 169.57, 170.04, 172.10.
Example 9
1,14-bis-tetradecanyl-5'-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-((-
.beta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine (20)
A solution of sodium methoxide (1M in methanol, 0.2 mL) was added
dropwise to a solution of compound 19 prepared in example 8 (0.64
g, 0.37 mmol) in 20 mL of methanol anhydrous. After heating for 30
minutes at 45.degree. C., Amberlite IRC-50 was added to convert
from Na.sup.+ to H.sup.+ ions. After 20 minutes at the same
temperature, the resin was removed by filtration and washed with
methanol. The filtrate was concentrated and the product was
purified by column chromatography on silica gel eluting with
MeOH:CH.sub.2Cl.sub.2 (30:70 to 50:50) to afford as a white solid
(Yield: 84%, 0.46 g).
.sup.1H NMR (300 MHz, MeOD) .delta. 1.22 (s, 20H), 1.48-1.57 (m,
4H), 1.87 (s, 6H), 2.11-2.16 (t, 4H), 2.22-2.27 (m, 4H), 3.41-3.51
(m, 6H), 3.64-3.70 (dd, 2H), 3.79-3.85 (m, 4H), 4.13-4.18 (m, 2H),
4.33-4.39 (m, 6H), 4.63-4.77 (m, 4H), 5.16 (s, 4H), 5.51-5.54 (d,
2H), 6.13-6.17 (t, 2H), 7.28 (s, 2H), 7.82 (s, 2H), 8.06 (s,
2H).
.sup.13C NMR (75 MHz, MeOD) .delta. 13.15, 26.89, 30.25, 30.38,
30.56, 30.66, 30.69, 35.57, 36.92, 37.06, 39.59, 52.73, 62.37,
70.87, 72.56, 73.94, 78.42, 81.13, 85.82, 88.31, 89.61, 111.15,
124.29, 125.34, 137.07, 144.58, 146.39, 151.95, 164.77, 176.27.
Example 10: Gelation Test
The aqueous dispersions (Milli-Q water, 18 M.OMEGA.cm.sup.-1)
containing 1.5 wt % of compounds 5 and 6 obtained in examples 1 and
2 were heated until dissolution (at 45.degree. C.) and gradually
allowed to cool to room temperature unless otherwise stated. The
resulting solid aggregate mass was stable to inversion of the
container when the test tube was turned upside down, which shows
that the compounds 5 and 6 form gels.
Example 11: Rheology
Rheological measurements were carried out on a Malvern Kinexus Pro+
rheometer with steel plate-plate geometry (20 mm diameter). The
lower plate is equipped with a Peltier temperature control system,
and all samples were studied at 25.+-.0.01.degree. C. unless
indicated otherwise. A solvent trap was used to ensure homogeneous
temperature and to prevent water evaporation. A gap distance of 0.3
mm was maintained between the plates. The gels based on compounds 5
(example 1) and 6 (example 2) (obtained in example were heated at
55.degree. C. and the liquid resulting was placed into the
rheometer and subjected to sinusoidal oscillations. All the
measurements were carried out within the linear viscoelastic regime
(LVR). For this purpose the experimental conditions to achieve a
linear viscoelastic regime were determined by performing an
amplitude strain sweep from 0.01 to 10% at an angular frequency of
1 Hz (6.283 rads.sup.-1).
FIG. 4 shows the frequency sweep results for hydrogels obtained
from compound 5 and 6 (at 23.10 mM) at a constant strain of
0.03%.
FIG. 5 shows the step-strain measurement of the hydrogel obtained
from compound 6 at 1% (w/v) at a fixed angular frequency of 6.283
rads.sup.-1. This experiment was repeated at least three times to
verify its reproducibility.
These results show the thixotropic behaviour of the hydrogel
according to the invention. The mechanical energy which is provided
destabilizes the gel, which is able to auto-regenerate when the
mechanical stress stops, without causing aging of the sample
(several stress/restauration of G' and G'' modulus were
observed).
Sol-Gel Transition Temperature (T.sub.gel)
The determination of gel-sol transition temperature can also be
determinated by rheology. The hydrogel (3% product 6 of example 2,
w/v) was heated progressively from 25 to 60.degree. C. (3.degree.
C./min). The oscillatory stress applied was set to 5 Pa at a
constant frequency (1 Hz). The sol-gel transition point was taken
as the temperature at which the gel became liquid. This melting
temperature is reached at the intersection of the viscoelastic
moduli (G' and G''). This value is of 46.degree. C. which is
compatible with the cell culture at the gel state (37.degree.
C.).
Example 12: Cytotoxicity and Cytocompatibility
The experiments were performed using compound 6 of example 2,
namely the
1,12-bis-dodecanyl-5'-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((.b-
eta.-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5'-deoxy
thymidine.
a) Cell Culture--Isolation and Culture of hASCs
Mesenchymal stem cells were isolated from human adipose tissue.
Human subcutaneous fat was obtained from healthy patients aged 20
to 80 years old who underwent hip surgery in Bordeaux Pellegrin CHU
(Bordeaux, France). Fat mass was separated from other tissues,
washed with sterilized PBS, finely cut and incubated with 0.1%
(w/v) collagenase type I (Worthington, Lakewood, N.J., USA) at
37.degree. C. with gentle agitation for 1 h30. After filtration and
centrifugation, the top liquid layer was removed and the remaining
Stromal Vascular Fraction (SVF) was treated for 10 min with ELB
(Erythocyte Lysis Buffer; 155 mM NH.sub.4Cl (Sigma-Aldrich, St.
Louis, Mo., USA), 5.7 mM K.sub.2HPO.sub.4, 7.4 mM
K.sub.2HPO.sub.4-3H.sub.2O, 0.1 mm EDTA (all Sigma-Aldrich)), and
then centrifuged. The pellet was resuspended in DMEM F12 medium
(Invitrogen/Life Technologies, Sergy Pontoise, France) supplemented
with 10% (v/v) Foetal Bovine Serum (FBS) (Lonza, Basel,
Switzerland) and sequentially filtered through 100, 70 and 40 .mu.m
cell strainer (BD Falcon, Franklin Lakes, N.J., USA). Cells were
plated and cultivated at 37.degree. C. in 5% CO.sub.2. Culture
medium was replaced every two days.
b) Cell Culture--D1-ORL-UVA
D1-ORL-UVA (ATCC.RTM. CRL-12424) were cultured in DMEM
(Invitrogen/Life Technologies) containing 10% FBS, and 1%
Penicillin-Streptomycin (Invitrogen/Life Technologies). Culture
medium was replaced every two days.
c) Preparation of 3D Scaffold with hASCs or D1 Seeding
For all in vitro assays, the hydrogel obtained from compound 6 was
prepared at a concentration of 3% (w/v). The compound 6 powder was
solubilized in PBS 1.times. under agitation (900 rpm) at 25.degree.
C. during 45 minutes. Then, when the gel was formed, it was left at
room temperature, without agitation, during 3 to 4 hours. To allow
cell seeding, the gel was heated during 30 minutes at 55.degree. C.
Once the liquid solution was obtained, it was rapidly cooled down
to 37.degree. C. and thoroughly mixed with 10 .mu.L cell suspension
at a concentration of 1 million cells per mL of gel. Finally, the
cellularized hydrogel was maintained under agitation (600 rpm), at
25.degree. C. during 45 minutes. After this period, medium was been
added (250 .mu.L/tube) and tubes were incubated at controlled
atmosphere (5% CO.sub.2, 37.degree. C.)
d) Evaluation of Compound 6 Cytotoxicity and of BOLA Gel
Cytocompatibility
The cytotoxicity of compound 6 was evaluated by the measurement of
the cell metabolic activity (MTT assay). This tests uses
tetrazolium bromide MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) which is reduced into formazan by the
mitochondrial enzyme succinate dehydrogenase of living cells. This
reduction results in a change of colour from yellow to violet-blue.
The colour intensity is proportional to the amount of living
cells.
Cells (hASCs or D1) were seeded in a 96 wells plate (Nunc) at a
density of 10 000 cells/cm.sup.2. Product 6 was solubilized at
concentrations from 5 .mu.M to 5 mM in culture media.
MTT assay was performed after 72 h of incubation. As a positive
control, the culture medium without GNBA was used. As a negative
control, Triton X100 was added in the culture medium. Results were
expressed as a percentage of the metabolic activity of the positive
control. Absorbance was measured at a wavelength of 570 nm with
background subtraction at 630 nm.
The cytotoxicity of compound 6 was determined by MTT test to
measure the metabolic activity of human mesenchymal stem cells
(ASCs) 48 hours after incubation with increasing concentrations of
compound 6. Results are shown as percentage of positive control
consisting in BOLA-free cell culture medium. **: p<0.01
(Mann-Whitney U-Test).
The results, represented on FIG. 6, show that compound 6 is non
toxic at concentrations up to approximately 1 mM.
Cell viability was also evaluated by using the Alamar Blue
metabolic activity assay. Resazurin sodium salt solution
(Sigma-Aldrich), suspended at a concentration of 0.1 mg/ml in PBS
1.times.), was diluted at 1/10 (v/v) in the culture media. The
solution was added to each well (250 .mu.L) and incubated during 3
h at 37.degree. C. After this time, the fluorescence was measured
at wavelengths excitation 530 nm and emission 590 nm. Triplicate
samples were analyzed for each experiment. The results are shown on
FIG. 7A.
Cytocompatibility was determined by monitoring alamar blue
metabolism in rat osteoblastic cells (A) or human ASCs (B) grown in
compound 6-based gels. Results are shown as percentage of the value
obtained at day 1. *: p<0.05; **: p<0.01 (Mann-Whitney
U-Test).
The results are shown on FIG. 7B.
Viability was assessed after 1, 4 and 7 days by using Live/Dead
staining with calcein-AM and ethidium homodimer (Molecular Probes,
Invitrogen, USA). Cells seeded within gels were incubated for 1 h
at 37.degree. C. in Hank's medium supplemented with 1.25 .mu.L
calcein-AM and 5 .mu.L EthD-1. Samples were observed with a
Confocal microscope (Leica TCS SPE).
Live/dead staining at 2 weeks showed a vast majority of green
(living) cells.
The results show that compound 6 is not cytotoxic and has a god
cytocompatibility when used as cell culture medium.
Example 13 (Comparative Example) Cytotoxycity of Compound GNL of G.
Godeau et al., Chem. Comm., 2009, 34, 5127-5130
The toxicity of the compound GNL disclosed in G. Godeau et al., on
page 5127, right column, was evaluated in human hepatic carcinoma
cells HuH-7 by MTT assay, in order to measure cellular
mortality.
Huh-7 cells were grown in DMEM medium supplemented with 10% foetal
calf serum, 2 mM L-glutamine and 1% non-essential amino acids, at
37.degree. C. in a 5% CO2 atmosphere. All culture reagents were
purchased from Invitrogen. 2000 Huh-7 cells per well were seeded
into a 96-well plate and incubated the following day with
increasing concentrations of compound GNL in complete growth
medium. After 5 days in the presence of the compounds, the living
cells were quantified by the colorimetric CellTiter Aqueous One
Solution Cell Proliferation Assay (Promega), as recommended.
The viability results are shown on FIG. 8.
The results show that the GNL compound does not have a significant
toxicity for concentrations which are equal to or lower than 100
.mu.M. For higher concentration, toxicity quickly becomes important
and 100% of the cells die at a concentration of 200 .mu.M.
* * * * *